This proposal explores the physiologic roles of stimulatory G protein (Gs) in bone, with an emphasis on interactions of this signaling system with parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP) receptor and Indian hedgehog (Ihh) signaling pathways. To assure physiologic relevance, mice are genetically manipulated to allow assessment in intact animals and tissues. Aim I will establish new ES cell lines missing one or both copies of the Gsalpha gene and generate chimeric mice containing both wild-type and Gsalpha mutant cells. The role of Gs in controlling cell proliferation will be assessed by standard histology and genomic Southern analysis in a survey of tissues. The possibility that Gs regulates differentiation of specific cell types will be assessed by standard histology and in situ hybridization. Except for bone, this is an initial survey, and detailed analysis of other tissues will involve collaborations beyond the scope of this proposal. Aim II will study the interactions of the PTH/PTHrP receptor and Gs signaling pathways in bone. Much of the PTH/PTHrP receptor signaling, which strongly regulates chondrocyte and osteoblast development, may use the Gs signaling pathway; other ligands may regulate Gs in bone as well. We will compare PTH/PTHrP receptor(-/-)/wild-type and Gsalpha (+/-) or Gsalpha (-/-)/wild-type chimeric mice as well as analyze PTH/PTHrP receptor(-/-)/Gsalpha (-/-) chimeric mice. Differentiation of chondrocytes and osteoblasts as well as phosphorylation of CREB protein, downstream of Gs, in these cells will be analyzed by standard histology, in situ hybridization and immunohistochemistry. Aim III will study the control of Ihh signaling by Gs in bone. In other settings, PKA, downstream of Gs, blocks Hedgehog action. Effects of deficient Gs signaling on expression of Ihh-target genes such as Patched (Ptc) and Gli in bone will be examined by in situ hybridization in Gsalpha (+/-) mice and Gsalpha (-/-)/wild-type chimeras. Together, these studies will define the interactions of Gs signaling with other important signaling pathways and the role of Gs in regulating normal bone development.